Trellis Bioscience has developed its patented CellSpot" technology into a robust platform for characterizing the secreted antibody from single human lymphocytes with regard to specificity and affinity against multiple antigens. For example, the platform was used to isolate a rare, high quality human antibody to Respiratory Syncytial Virus (RSV) that is now being prepared for human clinical testing by Trellis'licensee, MedImmune. In this project, we propose to use CellSpot to isolate therapeutic antibodies to combat Group A Streptococcus (GAS) infections. Blood lymphocytes from adults who have overcome GAS infection will be layered onto an antibody capture surface. After 5 hours of incubation, each cell's secreted antibody forms a "footprint" in the vicinity of the cell (~150 5m diameter). These footprints will then be incubated with fluorescent beads conjugated to the antigen target, which in this project will be the Shr protein of GAS. Shr is an iron transporter protein that is both evolutionarily conserved among GAS strains and required for survival in vivo. Spots that contain antibody to Shr will be detected under automated microscopic examination and the responsible cells will be isolated, followed by cDNA cloning of the antibody for further characterization following transfection into an expression system. To further insure that selected antibodies can detect Shr on the surface of GAS cells, fluorescent bacterial cells will also be included in the CellSpot analysis. These antibodies will be screened for physiological activity both by an in vitro opsonization assay to measure antibody-mediated bacteria killing by phagocytic blood cells and by an in vivo animal infection model to measure direct protection from infection. In Phase I of this project, we anticipate isolating ~15 human antibodies to the Shr protein, of which at least one will be shown to be protective in the animal model, confirming utility suggested by the protective nature of a rabbit polyclonal serum raised against Shr. Based on success in this Phase I objective, we will submit a Phase II application to advance the development of this antibody to clinical trial. The overall objective of this project is a therapeutic antibody to GAS which is well tolerated and highly effective in protecting against a broad range of GAS strains. PUBLIC HEALTH RELEVANCE: This project will use an innovative microscopic cell analysis technology to screen human lymphocytes for cells producing antibodies to Group A Streptococcus (GAS) Shr protein. The antibodies selected will have the ability to protect animals and, ultimately humans, during GAS infection and will be developed as new therapeutic agents for this disease.